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本帖最后由 qgjin 于 2010-5-7 04:30 编辑 1 G3 I1 h9 k: A, S& U2 d; A% Z
1 @4 L0 b1 Y. Y5 wPREPARATION OF MEF CULTURES2 t$ p) E. ~2 @. L6 M' ~9 x8 W
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard$ |9 q+ I1 Z6 K- a
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.( {4 R. c3 b+ T& `" ?' z
2. Mince the embryonic tissue in 5 ml of trypsin solution, transfer to an Erlenmeyer flask containing a stir bar and a few 5mm glass beads, and stir on& V- Y% o$ M3 G7 M3 s
magnetic stirrer for 15–25 min (use shorter incubation times if the embryos are E13 and use longer incubation times if the embryos are E16). Ideally,
( o( }4 |+ w0 V/ Q3 uthe resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
4 V" J: R2 p9 u0 J( [) }3. Pipette the cells with a 2-ml glass pipette to achieve cell suspension. Filter the suspension through a sieve or a screen, add 10 ml of7 W! Y4 k8 V, N
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.( {- T) Z# I; \1 @' B3 F
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
2 s/ N' b2 }7 }(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
5 a( n$ h, m2 k7 I0 G/ S5 L) S4. Plate the cell suspension onto cell culture plates at a density of about 2*106 cells per 100 mm plate (P0; i.e., passage 0) and incubate in
, N0 L$ Z+ a2 N- _1 G/ MMEF growth medium at 5% CO2 and 37℃ for 24 h.
2 ~# F7 {" D# z# v2 v0 E( _5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
5 Q$ g( l2 u) o; O6. Cultivate for an additional 1–2 d until the cells reach B90% confluence., W) L- u4 C7 v; q! h
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.$ l, X7 ^1 R$ x+ {. A
8. Add trypsin solution to the culture plates and incubate for 1–2 min.9 x2 Q. l9 |( Z
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split)./ J2 j4 F! G' Q# j0 V+ n' h1 E
10. Freeze any MEFs not needed immediately.% x4 A: s# w' P: e+ K' W; s% C5 N6 l
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
9 p/ o! w l& m4 U11. Continue to passage cells as described in Steps 6–9 of this Box. MEFs at passages 2–4 are most suitable as feeder layer for cultivation of* L& |9 R1 `, @* S M: X
undifferentiated maGSCs; prepare as follows.
. D1 R" U, V$ G% A" a) {12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h." g, U+ T3 P/ S& A1 r+ G4 F- Y
13. Aspirate the mitomycin C solution and wash three times with PBS.- w3 C3 z" F$ W1 [
14. Trypsinize MEFs as described in Steps 7–9 of this Box and replate them on new gelatin (0.1%)-treated microwell plates or to Petri dishes
6 P `6 x) J, O% D- xat a density of 50,000–60,000 cells/ cm2.
, x1 o2 ^3 A9 R- s& R -CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
. O" M; v- b1 M b- S3 I* Y3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
$ Q; `) S$ N& q7 v1 M2 ~passaging and thawing). |
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发表于 2010-5-7 04:20
发表于 2011-7-26 19:21
